A bioorthogonal chemistry strategy for probing protein lipidation in live cells

Abstract

We report a chemical lipidation model for the study of protein lipidations in vitro and in live mammalian cells based on a bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction.Protein lipidation-the covalent attachment of lipid anchors to a protein-regulates many cellular processes by controlling subcellular protein localization. 1 A prominent example is N-Ras, which controls cell growth, differentiation, and apoptosis by acting as a molecular switch in the signal transduction pathway, cycling between the inactive GDP-bound form and the active GTP-bound form. 2 As a prerequisite for its biological activity, N-Ras requires two sequential lipidations -a palmitoylation at Cys-181 and a farnesylation at Cys-186-in order to partition into proper compartments (Fig. 1a). 3 While the affinities of various lipid anchors toward membrane structures have been studied in vitro using the lipid bilayer model, 4 the dynamics of spatiotemporal segregation of proteins upon lipidation in vivo remains less well understood. 5 To probe the effect of lipidation on protein localization dynamics in vivo, two strategies have been successfully developed. One involved the fusion of fluorescent proteins to the target protein to facilitate its monitoring by confocal fluorescent microscopy. 6 Another involved the microinjection of the dye-labeled, semisynthesized proteins with the predetermined lipidation patterns into living cells followed by examination of its subcellular localizations by fluorescent microscopy. 7 While these two approaches have elucidated the effect of lipidation on protein localization, the spatiotemporal resolution of intracellular trafficking upon lipidation was limited. We have recently reported a photoinduced tetrazolealkene cycloaddition reaction for selective functionalization of either tetrazole or alkenecontaining proteins both in vitro and in vivo. 8 We envisioned that this photoinduced bioorthogonal reaction may provide a chemical strategy for probing protein lipidation in live cells with an improved spatiotemporal resolution. A two-step procedure will be employed in this approach; the photoreactive tetrazoles are first incorporated at the target lipidation sites, which then react with exogenous lipid dipolarophiles to form the lipidated products via the photoinduced cycloaddition reaction ( Correspondence to: Qing Lin, qinglin@buffalo.edu. Since successive lipidations are typically required for stable membrane association, we decided to examine the effect of lipid numbers by preparing two semi-synthetic EGFP proteins carrying varying number of photoreactive tetrazole moieties using the inteinmediated protein ligation strategy (Scheme 1). Thus, two short peptides containing either one (Tet 1) or two (Tet 2) tetrazole sidechains were prepared via solid-phase peptide synthesis. 10 In parallel, we cloned EGFP(1-239) into the ligation vector, pTXB1, and overexpressed EGFP-intein-CBD in E. coli. The ligation products, EGFP-Tet 3 and 4, were obtained by incubating the immobilized fusion prot...

Rapid, Photoactivatable Turn-On Fluorescent Probes Based on an Intramolecular Photoclick Reaction

“…13 Fluorescent pyrazoline cycloadducts were formed after the reactions, ensuring that only alkene- or tetrazole-pretagged proteins were labeled. Because the cycloaddition rate was modest (with k 2 values up to 10 M −1 s −1 ), relatively high concentrations of reagents (~100 µM) were typically required for labeling in the intracellular environment.…”

Shining a Light on Bioorthogonal Photochemistry for Polymer Science

“…The biorthogonality was demonstrated in vitro, by conjugation of green fluorescent proteins to alkene‐functionalized lipids, which were incorporated into live cells. [ 138 ] However, these systems were limited to in vitro experimentation, due to the short wavelength UV light necessary for activation.…”

Photo-Triggered Click Chemistry for Biological Applications

“…The photo-triggered cycloaddition reaction between EGFP-Tet and N -hexadecylmethacrylamide proceeded in bacterial lysate with ~52 % conversion based on LC–MS analysis. The application of the tetrazole-modified EGFP was also used to probe the effect of lipidation on protein localization in live cells without the use of lipidation enzymes [40]. …”